The earth's environment contains a multitude of microorganisms with which we are continuously interacting. The interactions can be beneficial, e.g. fermentations to produce wine, vinegar or antibiotics, neutral or even harmful as in the case of infectious diseases. The widespread presence of these microorganisms creates a continuing need for the detection, identification and study of their metabolic activity.
While the science of microbiology has changed significantly in the last quarter century, many procedures utilized for the detection, identification and analysis of the behavior of microorganisms are still time-consuming. For example, in the area of antimicrobic susceptibility, many of the hospitals in the United States still use tests which rely on the presence or absence of visible growth of microorganisms to indicate the efficacy of an antimicrobic compound. The most common of such tests is the Bauer-Kirby Disc Method which generally requires an 18 to 24 hour incubation period to allow for microorganism growth before a result can be obtained.
Another method of testing for antimicrobic susceptibility is the broth micro-dilution method, such as the Sceptor.RTM. System for identification and antimicrobic susceptibility testing of organisms (Becton Dickinson Diagnostic Instrumentation Systems, Sparks, Md.). The system uses a disposable plastic panel having a plurality of low volume cupulas (ca. 0.4 ml per cupula), each containing a different test compound or a different concentration of a test compound dried on the cupula surface. The organism to be tested is suspended in the desired testing medium, and aliquots are delivered to the individual cupulas of the test panel. The reagent dried on the panel dissolves in the sample, and the system is then incubated overnight (18 to 24 hours) to allow sufficient time for the organisms to interact with the reagent and for visible growth to appear. The panel is subsequently examined visually for the presence or absence of growth, thereby obtaining information on the susceptibility of the organism undergoing testing. Additional wells aid in identifying the organism. However, as indicated, this test method requires a long incubation period.
One approach to solving the requirement of long incubation periods is to monitor metabolic activity of the microorganisms rather than the growth of colonies. The growth of organisms in blood culture media can be monitored by a variety of methods such as detecting changes in turbidity, in pressure in a sealed culture vial, incorporation of radioactive substrates into metabolic products such as carbon dioxide, measuring the production of carbon dioxide or measuring the consumption of oxygen. As an example, apparatus with light scattering optical means have been used to ascertain susceptibility by determining the change in size or number of microorganisms in the presence of various antimicrobic compounds. Commercial instruments utilizing these principles are exemplified by the Vitec System (BioMerieux Corp.). This system claims to yield information on antimicrobic susceptibility of microorganisms within six hours for many organisms and drug combinations. Other combinations can require as long as 18 hours before the antimicrobic susceptibility of the organism can be determined by this machine.
Since the Bauer-Kirby procedure is still in use, modifications of this procedure have been developed which allow certain samples to be read in four to six hours. However, the modified system is "destructive"in nature, requiring the spraying of a developing solution of a color forming dye onto the test plate. Re-incubation and reading at a later time are, therefore, not possible and if the rapid technique fails, the experiment cannot be continued for a standard evaluation at a later time.
A bioluminescent method based on the quantity of adenosine triphosphate ("ATP") present in multiplying organisms has been described as yielding results of antimicrobic susceptibility testing in four and half hours for certain compositions (Wheat et al.). However, the procedure tends to be cumbersome and broad applicability has not been demonstrated.
Other approaches have involved monitoring microbial oxygen consumption by measuring pH and/or hemoglobin color change, or by using dyes such as triphenyl-tetrazolium chloride and resazurin, which change color in response to the total redox potential of the liquid test medium.
Monitoring the consumption of dissolved oxygen by microorganisms as a marker of their metabolism has been studied for many years. For example, C. E. Clifton monitored the oxygen consumption of microorganisms over a period of several days using a Warburg flask in 1937. This method measured the change in oxygen concentration in a slow and cumbersome manner.
The growth of microorganisms can also be monitored by the fluorescent output of a sensor deposited on the bottom of a blood culture vial, such as in the BACTEC.RTM. blood culture analyzer system (Becton Dickinson Diagnostic Instrumentation Systems, Sparks, Md.). Initially, the BACTEC.RTM. fluorescent blood culture analyzer system measured the production of carbon dioxide. Later, the BACTEC.RTM. system measured the consumption of oxygen.
The BACTEC.RTM. systems for measuring both carbon dioxide and oxygen are formulated using a silicone polymer as the sensor matrix to facilitate gas transmission through the sensor. Silicones are used because they are known to have one of the highest gas transmission of any synthetic polymer. The BACTEC.RTM. system detects oxygen consumption by the change of fluorescent output of a ruthenium compound having tris-(4,7-diphenyl-1,10-phenanthroline)ruthenium dichloride pentahydrate. This compound emits light (fluorescence) at a wavelength of 620 nanometers (NM) when excited by light at a wavelength of 440 NM. The fluorescence is stopped or quenched in the presence of oxygen. The detection of the microorganism is based on the theory that when a microorganism grows in a sealed culture vial, it will consume or deplete the oxygen inside the vial resulting in an increase in light output which is proportional to microbial growth.
The initial BACTEC.RTM. fluorescent blood culture analyzer system was developed by depositing tris-(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride pentahydrate from an ethanol solution onto the surface of silica gel. After the ethanol is removed, the resulting powder is compounded into a moisture curable liquid silicone polymer which is then added to a suitable container. Following curing (i.e. the liquid polymer is converted to a solid), culture media and headspace gases (e.g. carbon dioxide, oxygen and nitrogen) are added to the container which is capped.
The present BACTEC.RTM. fluorescent blood culture analyzer system requires the deposition of Tris-(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride pentahydrate from an ethanol solution on a silica gel. The coated silica gel is then mixed with silicone polymer in a heterogenous manner. The sensor thus produced is referred to as a two-phase or heterogeneous sensor. The coated silica gel will precipitate out of the silicone polymer mixture during processing unless mixing is provided. It has not been possible to directly add tris-(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride pentahydrate to the silicone to produce a functional sensor.
The required steps of depositing tris-(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride pentahydrate on silica gel from an ethanol solution and expansive mixing to prevent phase separation of coated silica from silicone polymer makes use of the heterogenous sensor expensive and time-consuming and not conducive to large-scale manufacturing.
Therefore, what is lacking in the art, and is now solved by the present invention, is a functional homogenous sensor which does not require the process step of depositing tris-(4,7-diphenyl-1,10-phenanthroline)ruthenium dichloride pentahydrate on silica gel from an ethanol.